Blebbistatin was discovered in a screen for chemical inhibitors of nonmuscle myosin IIA (NMIIA) ATPase activity. It inhibits blebbing and cytokinesis in Xenopus and human cells. We tested its activity against a number of other conventional and unconventional myosin isoforms. At low ATP concentrations (<100 micromolar) blebbistatin inhibited the actin-activated MgATPase activity of NMIIA HMM, NMIIB heavy meromyosin (HMM), smooth muscle HMM, and skeletal muscle HMM. The amount of blebbistatin required for half maximal inhibition of the actin-activated MgATPase is 0.5 and 50 micromolar for smooth muscle HMM. The actin-activated MgATPase of skeletal HMM was inhibited by 50% at 0.5 ?M blebbistatin. Blebbistatin, even at 100 micromolar, did not effectively inhibit the activity of myo1b, myosin-V and myosin-X. Blebbistatin completely inhibited the movement of rhodamine-phalloidin labeled F-actin by NMIIA and skeletal muscle HMM. This effect was reversible upon wash out of blebbistatin. At long times of observation (2-3 minutes) using rhodamine-excitation (532nm) in the presence of 100 micromolar blebbistatin, actin filaments would begin to move slowly in the field of observation. Subsequent movement of the slide to a new observation field would again show complete inhibition of motility suggesting that the effect was due to photochemical inactivation of blebbistatin and not a simple time dependent loss of inhibition. Alexa-488-excitation (488 nm) rapidly inactivated the blebbistatin such that no inhibition of movement of Alexa-488-phalloidin-labeled actin was apparent in the microscope. The latter experiment was conducted under evanescent illumination where the intensity of 488 nm light was extremely low, suggesting that blebbistatin is markedly sensitive to this wavelength. This property could be useful in studying the reversibility of blebbistatin inhibition of contractile processes in cells and for synchronizing contractile events.